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Processes
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State of the Art of Protein Expression Systems
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State of the Art of Protein Expression Systems

A special issue of Processes (ISSN 2227-9717). This special issue belongs to the section "Biological Processes and Systems".

Deadline for manuscript submissions: closed (20 September 2022) | Viewed by 19413

Special Issue Editors

Prof. Dr. Tzong-Yuan Wu

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Guest Editor
Department of Bioscience Technology, Chung Yuan Christian University, Chungli 320, Taiwan
Interests: baculovirus expression system; virology, protein physical chemistry
Prof. Dr. Monique M. van Oers

E-Mail Website
Guest Editor
Laboratory of Virology, Department of Plant Sciences, Wageningen University, Wageningen, The Netherlands
Interests: insect viruses; virus-host interactions; baculovirus; virus evolution; virus taxonomy; antiviral defense; caterpillars; bees; dipteran insects
Special Issues, Collections and Topics in MDPI journals
Dr. Li-Fen Huang

E-Mail Website
Guest Editor
Graduate School of Biotechnology and Bioengineering, Yuan Ze University, 135 Yuan-Tung Road, Chung-Li 320, Taiwan
Interests: engineering protein expression system in rice cells for valuable medical protein production; DNA detection biosensor for GMO; Develop plant tissue culture systems for Chinese medical plants, snow lotus, snow lotus

Special Issue Information

Dear Colleagues,

One of the big social impacts of biotechnology is the production of recombinant proteins. Recombinant proteins have dramatically changed the art of medical treatments, drug discoveries, vaccine developments, as well as basic biological research studies. However, recombinant protein production is the basic challenge for ongoing medical applications and basic research studies. Thus, the protein expression system, as an interesting and fast-growing research area in biotechnology, might be an attractive tool to solve this challenge.

The Special Issue “State of the Art of Protein Expression System” covers original research papers or reviews on protein expression systems, e.g., Escherichia coli, Bacillus subtilis, Lactococcus lactis, yeast, insect cells, mammalian cells, animal, as well as plants.

This Special Issue aims to focus on recent progresses and advances in vectors design, secretion signals, recombinant protein purifications, oligomeric protein expression, virus-like particle production and subunit vaccines production.

Topics include, but are not limited to:

  • vector designs for protein expression system;
  • investigations on signal peptide for recombinant proteins production;
  • downstream process of recombinant proteins;
  • development of oligomeric recombinant expression vectors;
  • virus like particle production and preparations;
  • subunit vaccines production and preparations.

Prof. Dr. Tzong-Yuan Wu
Prof. Dr. Monique M. van Oers
Dr. Li-Fen Huang
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Processes is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2400 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • downstream process
  • protein expression system
  • recombinant proteins
  • vectors design
  • signal peptide
  • vaccine
  • virus-like particle

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Published Papers (7 papers)

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Research

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16 pages, 3747 KiB  
Article
Sortase A Fusion Expression and mIFc2 Co-Expression of Bovine Lactoferricin and Analysis of Its Antibacterial Activity
by Chao-Yu Hsu, Chung-Yiu Hsieh, Cheng-Yao Yang, Yu-Kang Chang, Wen-Ling Shih, Chuan-Ming Yeh, Nien-Jen Hu, Ming-Shan Chen, Brent L. Nielsen and Hung-Jen Liu
Processes 2022, 10(12), 2470; https://doi.org/10.3390/pr10122470 - 22 Nov 2022
Viewed by 1655
Abstract
The coding region for the sortase A (SrtA) of Staphylococcus aureus was fused at the N-terminus of LfcinB. The SrtA-LfcinB fusion protein in E. coli C43(DE3) was expressed with the expected sizes of 21 kDa and 38 kDa by pET21b-SrtA-LfcinB and pET32-1SrtA-LfcinB constructs, [...] Read more.
The coding region for the sortase A (SrtA) of Staphylococcus aureus was fused at the N-terminus of LfcinB. The SrtA-LfcinB fusion protein in E. coli C43(DE3) was expressed with the expected sizes of 21 kDa and 38 kDa by pET21b-SrtA-LfcinB and pET32-1SrtA-LfcinB constructs, respectively. Increased levels of the TrxA-His-SrtA-SrtA-LfcinB fusion protein were detected by the pET32-3SrtA-LfcinB construct having three expression cassettes. LfcinB is released from the expressed SrtA-LfcinB protein by SrtA self-cleavage which is induced in the presence of Ca2+. The antibacterial activity was detected after SrtA-mediated cleavage of LfcinB. Furthermore, to reduce the antimicrobial peptide toxicity to the E. coli host, the human interferon-γ (hIFN-γ) sequences were mutated into a negatively charged mIFc2 protein (7 kDa), which was co-expressed with LfcinB in an insoluble form. The yield of LfcinB was elevated while changing the gene order of LfcinB and mIFc2 (pET21b-fLfcinB-bmIFc2). Furthermore, increased levels of LfcinB were detected using the pET21b-(fLfcinB-bmIFc2)2 construct. To increase the dissolution rate of inclusion bodies, inclusion bodies treated with different temperatures and pH and resuspended in different volumes of 50 mM Tris-HCl were assayed. Our results reveal that heat-treated LfcinB/mIFc2 inclusion bodies at 90 °C, pH 10, and 16X resuspended volumes have the best resolubilization rate. This work suggests that the mIFc2 co-expression system shows higher efficiency for LfcinB production than the SrtA fusion system. The expressed LfcinB from the mIFc2 co-expression system exhibits excellent broad-spectrum antibacterial activities against thirteen Gram-negative and ten Gram-positive bacteria species with a range of minimum inhibitory concentrations (MIC) between 37–150 ug/mL. Full article
(This article belongs to the Special Issue State of the Art of Protein Expression Systems)
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16 pages, 2129 KiB  
Article
Targeted Metabolic Analysis and MFA of Insect Cells Expressing Influenza HA-VLP
by Alexandre B. Murad, Marcos Q. Sousa, Ricardo Correia, Inês A. Isidro, Manuel J. T. Carrondo and António Roldão
Processes 2022, 10(11), 2283; https://doi.org/10.3390/pr10112283 - 4 Nov 2022
Cited by 1 | Viewed by 1658
Abstract
Virus-like particles (VLPs) are versatile vaccine carriers for conferring broad protection against influenza by enabling high-level display of multiple hemagglutinin (HA) strains within the same particle construct. The insect cell-baculovirus expression vector system (IC-BEVS) is amongst the most suitable platforms for VLP expression; [...] Read more.
Virus-like particles (VLPs) are versatile vaccine carriers for conferring broad protection against influenza by enabling high-level display of multiple hemagglutinin (HA) strains within the same particle construct. The insect cell-baculovirus expression vector system (IC-BEVS) is amongst the most suitable platforms for VLP expression; however, productivities vary greatly with particle complexity (i.e., valency) and the HA strain(s) to be expressed. Understanding the metabolic signatures of insect cells producing different HA-VLPs could help dissect the factors contributing to such fluctuations. In this study, the metabolic traces of insect cells during production of HA-VLPs with different valences and comprising HA strains from different groups/subtypes were assessed using targeted metabolic analysis and metabolic flux analysis. A total of 27 different HA-VLP variants were initially expressed, with titers varying from 32 to 512 HA titer/mL. Metabolic analysis of cells during the production of a subset of HA-VLPs distinct for each category (i.e., group 1 vs. 2, monovalent vs. multivalent) revealed that (i) expression of group-2 VLPs is more challenging than for group-1 ones; (ii) higher metabolic rates are not correlated with higher VLP expression; and (iii) specific metabolites (besides glucose and glutamine) are critical for central carbon metabolism during VLPs expression, e.g., asparagine, serine, glycine, and leucine. Principal component analysis of specific production/consumption rates suggests that HA group/subtype, rather than VLP valency, is the driving factor leading to differences during influenza HA-VLPs production. Nonetheless, no apparent correlation between a given metabolic footprint and expression of specific HA variant and/or VLP design could be derived. Overall, this work gives insights on the metabolic profile of insect High Five cells during the production of different HA-VLPs variants and highlights the importance of understanding the metabolic mechanisms that may play a role on this system’s productivity. Full article
(This article belongs to the Special Issue State of the Art of Protein Expression Systems)
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11 pages, 706 KiB  
Communication
Plum Pox Virus Genome-Based Vector Enables the Expression of Different Heterologous Polypeptides in Nicotiana benthamiana Plants
by Adam Achs, Miroslav Glasa and Zdeno Šubr
Processes 2022, 10(8), 1526; https://doi.org/10.3390/pr10081526 - 3 Aug 2022
Cited by 2 | Viewed by 1706
Abstract
Plant viral vectors have become a promising tool for the rapid and cost-effective production of recombinant proteins in plants. Among the numerous genera of viruses that have been used for heterologous expression, potyviruses offer several advantages, such as polyprotein expression strategy or a [...] Read more.
Plant viral vectors have become a promising tool for the rapid and cost-effective production of recombinant proteins in plants. Among the numerous genera of viruses that have been used for heterologous expression, potyviruses offer several advantages, such as polyprotein expression strategy or a broad host range. In our work, the expression vectors pAD/pAD-agro based on the plum pox virus (PPV) genome were used for the heterologous expression of different foreign polypeptides: alfalfa mosaic virus capsid protein (AMV CP), zucchini yellow mosaic virus capsid protein (ZYMV CP), the small heat-shock protein of Cronobacter sakazakii fused with hexahistidine (sHSP-his), a fragment of influenza A virus hemagglutinin (HA2-2), influenza A virus protein PB1-F2, SARS-CoV-2 nucleocapsid protein (CoN2-his), and its N- and C-terminal fragments (CoN-1-his and CoN3-his, respectively), each fused with a hexahistidine anchor. Particular proteins differed in their accumulation, tissue localization, stability, and solubility. The accumulation rate of produced polypeptides varied from low (N, hemagglutinin fragment) to relatively high (plant viral CPs, N-terminal fragment of N, PB1-F2). Some proteins preferentially accumulated in roots (sHSP, hemagglutinin fragment, PB1-F2), showing signs of proteolytic degradation in leaf tissues. Thus, each expression requires an individual approach and optimization. Here, we summarize our several-year experiments and discuss the usefulness of the pAD/pADep vector system. Full article
(This article belongs to the Special Issue State of the Art of Protein Expression Systems)
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13 pages, 2380 KiB  
Article
Secreted Trimeric Chikungunya Virus Spikes from Insect Cells: Production, Purification, and Glycosylation Status
by Tessy A. H. Hick, Corinne Geertsema, Maurice G. L. Henquet, Dirk E. Martens, Stefan W. Metz and Gorben P. Pijlman
Processes 2022, 10(1), 162; https://doi.org/10.3390/pr10010162 - 14 Jan 2022
Cited by 1 | Viewed by 3054
Abstract
Chikungunya virus (CHIKV) is a rapidly emerging mosquito-borne virus that causes a severe febrile illness with long-lasting arthralgia in humans. As there is no vaccine to protect humans and limit CHIKV epidemics, the virus continues to be a global public health concern. The [...] Read more.
Chikungunya virus (CHIKV) is a rapidly emerging mosquito-borne virus that causes a severe febrile illness with long-lasting arthralgia in humans. As there is no vaccine to protect humans and limit CHIKV epidemics, the virus continues to be a global public health concern. The CHIKV envelope glycoproteins E1 and E2 are important immunogens; therefore, the aim of this study is to produce trimeric CHIKV spikes in insect cells using the baculovirus expression system. The CHIKV E1 and E2 ectodomains were covalently coupled by a flexible linker that replaces the 6K transmembrane protein. The C-terminal E1 transmembrane was replaced by a Strep-tag II for the purification of secreted spikes from the culture fluid. After production in Sf9 suspension cells (product yields of 5.8–7.6 mg/L), the CHIKV spikes were purified by Strep-Tactin affinity chromatography, which successfully cleared the co-produced baculoviruses. Bis(sulfosuccinimidyl)suberate cross-linking demonstrated that the spikes are secreted as trimers. PNGase F treatment showed that the spikes are glycosylated. LC–MS/MS-based glycoproteomic analysis confirmed the glycosylation and revealed that the majority are of the mannose- or hybrid-type N-glycans and <2% have complex-type N-glycans. The LC –MS/MS analysis also revealed three O-glycosylation sites in E1. In conclusion, the trimeric, glycosylated CHIKV spikes have been successfully produced in insect cells and are now available for vaccination studies. Full article
(This article belongs to the Special Issue State of the Art of Protein Expression Systems)
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13 pages, 3182 KiB  
Article
Optimizing Recombinant Baculovirus Vector Design for Protein Production in Insect Cells
by Carina Bannach, Daniel Ruiz Buck, Genna Bobby, Leo P. Graves, Sainan Li, Adam C. Chambers, Elizabeth Gan, Raquel Arinto-Garcia, Robert D. Possee and Linda A. King
Processes 2021, 9(12), 2118; https://doi.org/10.3390/pr9122118 - 25 Nov 2021
Cited by 2 | Viewed by 3915
Abstract
Autographa californica nucleopolyhedrovirus is a very productive expression vector for recombinant proteins in insect cells. Most vectors are based on the polyhedrin gene promoter, which comprises a TAAG transcription initiation motif flanked by 20 base pairs upstream and 47 base pairs downstream before [...] Read more.
Autographa californica nucleopolyhedrovirus is a very productive expression vector for recombinant proteins in insect cells. Most vectors are based on the polyhedrin gene promoter, which comprises a TAAG transcription initiation motif flanked by 20 base pairs upstream and 47 base pairs downstream before the native ATG. Many transfer vectors also include a short sequence downstream of the ATG, in which case this sequence is mutated to ATT to abolish translation. However, the ATT sequence, or AUU in the mRNA, is known to be leaky. If a target-coding region is placed in the frame with the AUU, then some products will comprise a chimeric molecule with part of the polyhedrin protein. In this study, we showed that if AUU is placed in the frame with a Strep tag and eGFP coding region, we could identify a protein product with both sequences present. Further work examined if alternative codons in lieu of AUG might reduce translation initiation further. We found that AUA was used slightly more efficiently than AUU, whereas AUC was the least efficient at initiating translation. The use of this latter codon suggested that there might also be a slight improvement of protein yield if this is incorporated into expression vectors. Full article
(This article belongs to the Special Issue State of the Art of Protein Expression Systems)
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Review

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14 pages, 887 KiB  
Review
Dunaliella salina as a Potential Biofactory for Antigens and Vehicle for Mucosal Application
by Inkar Castellanos-Huerta, Gabriela Gómez-Verduzco, Guillermo Tellez-Isaias, Guadalupe Ayora-Talavera, Bernardo Bañuelos-Hernández, Víctor Manuel Petrone-García, Isidro Fernández-Siurob, Luis Alberto Garcia-Casillas and Gilberto Velázquez-Juárez
Processes 2022, 10(9), 1776; https://doi.org/10.3390/pr10091776 - 4 Sep 2022
Cited by 6 | Viewed by 3641
Abstract
The demand for effective, low-cost vaccines increases research in next-generation biomanufacturing platforms and the study of new vaccine delivery systems (e.g., mucosal vaccines). Applied biotechnology in antigen production guides research toward developing genetic modification techniques in different biological models to achieve the expression [...] Read more.
The demand for effective, low-cost vaccines increases research in next-generation biomanufacturing platforms and the study of new vaccine delivery systems (e.g., mucosal vaccines). Applied biotechnology in antigen production guides research toward developing genetic modification techniques in different biological models to achieve the expression of heterologous proteins. These studies are based on various transformation protocols, applied in prokaryotic systems such as Escherichia coli to eukaryotic models such as yeasts, insect cell cultures, animals, and plants, including a particular type of photosynthetic organisms: microalgae, demonstrating the feasibility of recombinant protein expression in these biological models. Microalgae are one of the recombinant protein expression models with the most significant potential and studies in the last decade. Unicellular photosynthetic organisms are widely diverse with biological and growth-specific characteristics. Some examples of the species with commercial interest are Chlamydomonas, Botryococcus, Chlorella, Dunaliella, Haematococcus, and Spirulina. The production of microalgae species at an industrial level through specialized equipment for this purpose allows for proposing microalgae as a basis for producing recombinant proteins at a commercial level. A specie with a particular interest in biotechnology application due to growth characteristics, composition, and protein production capacity is D. salina, which can be cultivated under industrial standards to obtain βcarotene of high interest to humans. D saline currently has advantages over other microalgae species, such as its growth in culture media with a high salt concentration which reduces the risk of contamination, rapid growth, generally considered safe (GRAS), recombinant protein biofactory, and a possible delivery vehicle for mucosal application. This review discusses the status of microalgae D. salina as a platform of expression of recombinant production for its potential mucosal application as a vaccine delivery system, taking an advance on the technology for its production and cultivation at an industrial level. Full article
(This article belongs to the Special Issue State of the Art of Protein Expression Systems)
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12 pages, 1652 KiB  
Review
Current Strategies to Improve Yield of Recombinant Protein Production in Rice Suspension Cells
by Yu-Hsiang Chi and Li-Fen Huang
Processes 2022, 10(6), 1120; https://doi.org/10.3390/pr10061120 - 3 Jun 2022
Cited by 6 | Viewed by 5825
Abstract
A plant cell-based recombinant glucocerebrosidase was approved by the FDA in 2012 for the treatment of human inherited Gaucher disease, indicating that plant suspension cells have advantages in biosafety and a low production cost as a commercial pharmaceutical recombinant protein expression system. A [...] Read more.
A plant cell-based recombinant glucocerebrosidase was approved by the FDA in 2012 for the treatment of human inherited Gaucher disease, indicating that plant suspension cells have advantages in biosafety and a low production cost as a commercial pharmaceutical recombinant protein expression system. A low allergenic rice suspension cell-based recombinant protein expression system controlled by the αAmy3/RAmy3D promoter has been shown to result in relatively high protein yields in plant cell-based systems. Although several recombinant proteins have been produced in rice suspension cell-based systems, yields must be improved to compete with the current commercial protein expression systems. Different strategies were performed and showed successful improvements in recombinant protein yields in this rice system. The review updates and highlights strategies for potential improvements of the αAmy3-based rice suspension cell-based system. Full article
(This article belongs to the Special Issue State of the Art of Protein Expression Systems)
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